THE  PERMEABILITY 


OF 

THE  SEED  COAT  OF  PEAS 


BY 


SIEGFRIED  PAUL  HARTER 


THESIS 


FOR  THE 

DEGREE  OF  BACHELOR  OF  ARTS 


IN 


BOTANY 


COLLEGE  OF  LIBERAL  ARTS  AND  SCIENCES 
UNIVERSITY  OF  ILLINOIS 


1922 


/ 922 
h)25 


UNIVERSITY  OF  ILLINOIS 


Ma_£_  25j_ 


THIS  IS  TO  CERTIFY  THAT  THE  THESIS  PREPARED  UNDER  MY  SUPERVISION  BY 
Si  e Lgfr  i ed_  _Paul_  _Har  te  r 

ENTITLED T-he-  1-i  ty  - -of  - -th-e  - -S  ee-d-  -G  ©-at- -q£-  - P-e  a© 


IS  APPROVED  BY  ME  AS  FULFILLING  THIS  PART  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF  Bachelor  of  Arts  in  Botany 


Instructor  in  Charge 


4 


HEAD  OF  DEPARTMENT  OF  __J?j)tany_ 


Digitized  by  the  Internet  Archive 
in  2015 


https://archive.org/details/permeabilityofseOOhart 


1 


Table  of  Contents. 

I  Introduction. 

II  Citation  of  References. 

Ill  Methods  and  Materials. 

IV  Results  and  Experimental  Data. 

V  Discussion  and  Interpretation. 

VI  Conclusions. 

VII  Figures. 


2 


I Introduction. 

It  has  long  been  known  that  dry  seeds,  spores,  and  other 
dormant  stages  of  plants  possess  an  enormous  resistance  to 
very  poisonous  substances.  The  early  investigations  along 
this  line  were  carried  on  principally  with  the  weaker  solu- 
tions. More  recently  a great  deal  of  work  has  been  done  with 
the  higher  concentrations  of  acids  and  salt  solutions,  with 
especial  reference  to  their  effect  upon  the  dry  seeds  of  the 
leguminous  plants.  Many  of  these  investigators  have  reported 
certain  maxima  of  poisonous  effect  at  certain  concentrations 
of  the  toxic  substances.  One  of  the  earliest  of  these  exper- 
iments was  carried  on  by  V.  Arcichovskij,  and  his  results  are 
set  forth  in  an  article  in  Biochem.  Zeitschrift  Vol.  50,  233, 

«<  ft 

entitled  "Biochemi sche  wirkung  hochst  Konzentrierter  Losungen". 
His  results  are  especially  interesting  here  because  his  work 
forms  the  basis  for  a considerable  portion  of  this  investigation. 

Arcichovskij  carried  on  his  work  with  the  seeds  of  peas, 
but  the  particular  variety  of  seeds  which  he  used  is  not  men- 
tioned. In  the  course  of  his  experiments  he  treated  seeds 
with  Formalin,  Sulfuric  acid,  and  ITitric  acid;  using  varying 
concentrations  and  a varying  period  off  exposure.  Elaborate 
precautions  were  taken  against  bacterial  contamination  of  the 
germinating  seeds  and  a very  scrupulous  system  of  washing  the 
treated  seeds  was  resorted  to.  The  greater  part  of  his  work 
was  carried  on  with  formalin  and  he  concluded  for  this  poison 
that  the  stronger  concentrations  of  formalin  even  after  a 
long  periods  of  immersion  showed  relatively  small  poisonous 


3 


effect,  whereas  concentrations  of  medium  strength  gave  maxi- 

2 

mum  killing.  The  highest  poisonousness  was  evinced  by  the 
Q%  formalin. 

In  his  tests  with  Sulfuric  Acid  he  used  a 2 hour  imer- 
sion  in  all  tests  and  varied  the  concentrations  from  n/128 
to  concentrated.  The  table  of  results,  as  he  gives  it, 
follows: 

Cone.  H2S04  n/128  n/32  n/8  n/4  n/2  n/l  2n  4n  8n  16n  32n 
% of  germ  94  92  76  48  24  49.5  24.5  92  100  100  100 

Cone. 

100. 

As  in  the  case  of  the  work  with  formalin  it  will  be  observed 
that  a higher  percent  of  germination  was  obtained  with  the 
stronger  concentrations  of  acids.  Here  a maximum  killing  was 
obtained  at  l/2n  solution.  He  further  found  that  some  seeds 
will  withstand  a 16  hour  storage  in  concentrated  H2S04. 

With  Nitric  acid  a maximum  poi sonousness  was  obtained 
with  a normal  solution  and  a 2 hour  treatment.  The  general 
conclusion  from  the  work  was  that  the  resistance  of  seeds  to 
poisons  depends  upon  the  physical  properties  of  the  solutions 
and  that  the  strongest  concentrations  in  all  cases  are  the 
least  poisonous. 

A more  recent  study  of  the  same  problem  is  that  of  A.  J. 
Brown.  This  work  was  conducted  on  the  seeds  of  Horedum  vul- 
gare,  and  the  results  are  reported  in  an  article  entitled 
n0n  the  Existence  of  a Semipermeable  Membrane  enclosing  the 
Seeds  of  some  Gramineae. " Here  too,  various  concentrations 


« 


* 


. 


- 


- 

- 


, 


. 


« 

4 

of  H2S04  were  used,  together  with  various  lengths  of  treat- 
ment. In  the  particular  variety,  caerulescens,  which  was 
used,  the  grain  contains  a blue  pignent  in  the  aleurone  cells 
which  turns  to  red  with  acid,  thus  making  it  a valuable  in- 
dicator. Brown  found  a marked  difference  in  the  behavior  of 
damaged  seeds  and  those  which  were  intact.  The  damaged  seeds 
changed  color  at  once  in  even  a 1 $ solution  of  H2S04,  whereas 
in  the  case  of  perfect  seeds  no  color  change  resulted  even 
after  a week’s  treatment  in  such  a solution.  In  this  case, 
however,  swelling  took  place  at  once,  thus  indicating  that 
water  had  entered  the  seed.  Sections  cut  longitudinally  and 
cross  wise  through  such  seeds  also  show  no  trace  of  the  en- 
trance of  acid,  but  give  abundant  evidence  of  the  admission  of 
water.  By  titrating  acid  solutions  before  and  after  steeping 
seeds  in  them  it  was  also  found  that  undamaged  seeds  abstract 
water  from  H2S04  solutions  not  exceeding  4*9$. 

In  working  with  more  concentrated  solutions  9,  18,  and 
36$  H2S04  for  44  hour  periods  of  treatment  no  acid  was  ab- 
sorbed in  any  case  by  the  undamaged  seeds,  and  the  amount  of 
water  absorbed  decreases  inversely  as  the  concentration  of  the 
acid.  Good  germination  was  secured,  after  washing,  with  treat- 
ment in  18  to  36$  solutions.  The  same  selective  action  was 
manifested  for  HN03  and  Iodine,  although  HN03  proved,  by  first 
boiling  the  undamaged  grains  and  then  treating  with  acid,  that 
this  semi-permeability  is  not  a function  of  the  living  proto- 
plasm, but  is  resident  in  the  seed  coat.  An  attempt  was  also 
made  to  find  the  particular  portion  of  the  seed  coat  which 


' 


, 

" 

, - 


5 


was  responsible  for  the  semi-perm eabl e property. 

Brown  came  to  several  very  definite  and  significant  con- 
clusions. He  found  that  the  grain  of  Hordeum,  Avena,  Triticum, 
and  Secale,  is  enclosed  in  a semi-permeabl e covering  which 
permits  the  passage  of  water  and  iodine  to  the  interior  of  the 
grain,  but  prevents  the  passage  of  H2S04,  HC1 , and  all  salts 
of  metals  thus  far  examined,  when  they  are  in  aqueous  solution. 
This  semi -permeable  or  selective  property  is  located  someplace 
in  the  sperraoderm  of  the  seed. 

It  would  appear  that  a large  proportion  of  the  work  which 
has  already  been  done  in  connection  with  the  problem  is  more 
or  less  irrelevant  to  the  real  bearings  of  the  question. 
Arcichovskij  placed  an  undue  emphasis  upon  uselessly  elabor- 
ate washing  and  sterilization  methods,  whereas  he  failed  to 
be  rigorous  enough  in  his  germination  criterion.  Many  seeds 
which  he  would  count  as  germinated  are  in  reality  damaged  be- 
yond viability. 

His  general  conclusions  that  the  poisons  affected  the 
germination  of  the  seeds  to  a greater  or  lesser  extent,  and 
that  thereby  resistance  to  microorgainisms  was  weakened,  fail 
to  give  any  explanation  for  the  difficulty.  He  admits  that 
the  reasons  for  the  lessened  poisonous  effect  of  higher  con- 
centrations are  probably  very  complex.  It  is  also  quite 
apparent  that  the  dry  protoplasmic  state  of  the  seeds  causes 
them  to  be  particularly  resistant,  and  that  the  chemical  ac- 
tivity of  substances  in  higher  concentrations  is  much  less 
than  in  weaker  ones.  Like  Kahlenberg  and  True,  Arcichovskij 


* 


6 


lays  stress  on  the  fact  that  in  the  case  of  electrolytes, 
stronger  solutions  are  relatively  and  absolutely  less  disso- 
ciated. 

H.  Schroeder,  on  the  other  hand,  in  his  article  in  Slora 

ii 

Vol.  102,  p 186,  "liber  die  Selektiv  permeabele  Hulle  des 
Weizenkornes"  concludes  that  peas  do  not  possess  a selective 
permeable  membrane.  He  finds  that  peas  manifest  no  difference 
in  absorption  rate  when  immersed  in  increasing  concentrations, 
nor  is  there  any  difference  in  absorption  rate  between  injured 
and  uninjured  seeds.  He  points  out  that  any  change  in  the  con- 
centration of  the  surrounding  solution  in  the  case  of  pea  seeds 
is  of  only  temporary  significance. 

In  a Cornell  University  bulletin  entitled  " Germination  of 
Seed  as  affected  by  Sulfuric  Acid  Treatment",  Love  and  Leighty 
attempt  to  make  a practical  use  of  the  results  of  experimental 
investigations  of  the  cause  of  hard  seeds.  In  using  varieties 
of  clover,  alfalfa,  sweet  clover,  weed  seeds,  and  cotton  seeds, 
they  got  results  which  were  too  inconsistent  to  be  of  great 
practical  value.  They  did,  however,  demonstrate  the  fact  that 
the  hard  leguminous  seeds  show  an  increase  in  the  percentage 
and  the  rate  of  gemination  after  treatment  with  concentrated 
H2S04  for  varying  lengths  of  time,  and  that  in  spite  of  the 
fact  that  there  was  great  individual  variation  in  regard  to 
hardness,  there  is  a certain  maximum  period  of  treatment  with 
which  damage  results  to  the  seed.  A period  of  treatement  ex- 
ceeding two  hours  proved  dangerous  in  most  cases. 


*. 


. 


7 


Work  of  a somewhat  similar  nature  was  done  by  Bergtheil 
and  Bay  in  an  article,  "On  the  Cause  of  Hardness  in  the  Seeds 
of  Indigofera  aurrecta  in  Annals  of  Botany?  Vol.  21,  p 57, 
they  report  the  results  of  their  investigations.  They  appear 
to  credit  the  original  use  of  H2S04  as  a scarifying  agent  to 
hasten  the  germination  of  seeds  to  the  studies  of  Dr.  Butler 
on  treating  cotton  seeds  for  the  destruction  of  insect  eggs. 
They  found  that  after  only  a 20  minute  to  30  minute  treatment 
with  concentrated  H2S04,  and  consequent  washing  and  drying, 
the  germination  is  perfect.  They  point  out  also  that  the 
reasons  for  such  an  increased  germination  after  treatment  wi  tli 
H2S04  were  alleged  by  Percival  to  be  due  to  the  fact  that  the 
testae  contain  a relatively  large  ash  constituent  and  hence 
resist  the  penetration  of  water  until  they  are  scarified  by 
the  action  of  the  acid# 

This  explanation,  however,  according  to  Bergtheil  and 
Day  is  not  sufficient  in  the  case  under  their  observation,  viz. 
that  of  Indigofera  aurrecta.  They  proceed  in  turn  to  attempt 
an  explanation  of  the  impermeability  by  studying  the  structur e 
or  organic  constituents  of  the  3eed  coat.  By  the  use  of  stain- 
ing material  it  was  decided  that  the  impermeable  layer  is  on 
the  extreme  outside  of  the  seed  coat,  and  the  conclusion  ar- 
rived at  was  "that  the  hard  nature  of  the  seeds  of  Indigofera 
aurrecta  is  due  to  their  possession  of  a very  thin  outer  cover- 
ing of  a substance  resistant  to  water-pro bably  a transition 
product  between  cellulose  abd  cuticle.  Scarifying  removes  a 
portion  of  this  resistant  covering  and  so  allows  penetration 


. 


8 


of  water,  while  H2S04  does  the  same  by  causing  rupturing  and 
swelling  or  by  converting  it  into  a body  akin  to  cellulose 
and  hence  permeable  to  water. 

Then,  directly  in  keeping  with  these  findings,  Paine 
and  Saunders,  while  experimenting  with  sources  of  material 
which  would  give  a non-extensible  membrane  for  a Pfeffer’s 
cell,  found  what  they  considered  to  be  the  solution  for  the 
problem.  In  an  article,  "On  a Peculiarity  Exhibited  by  the 
Testa  of  Wrinkled  Peas"  in  Annals  of  Botany,  Vol.  32,  p 175, 
they  maintain  that  the  differences  in  permeability  exhibited 
by  the  seeds  coats  of  peas  is  due  to  a layer  of  waxy  bloom 
on  the  surface  of  the  seed.  This  conclusion  was  arrived  at 
after  it  had  been  discovered  that  there  was  a marked  differ- 
ence in  the  permeability  of  the  seed  coats  of  smooth  and 
wrinkled  peas.  With  smooth  peas  a uniform  peimeability  was 
obtained,  whereas  with  wrinkled  ones  a very  irregular  and 
much  netted  permeability  was  evidenced.  Upon  further  inves- 
tigation it  was  found  that  the  depressions  in  the  coats  of 
wrinkled  peas  were  impermeable  while  the  ridges  were  permeable 
to  a UaCl  solution.  Upon  soaking  in  warm  alcohol  for  several 
hours  the  permeability  was  perfectly  uniform.  It  was  decided 
that  in  the  case  of  the  smooth  pea  the  bloom  layer  was  readily 
rubbed  off,  whereas,  with  a wrinkled  pea  the  wax  would  be 
rubbed  only  from  the  ridges,  thus  rendering  thorn  permeable. 

How  as  a result  of  this  vast  amount  of  work  on  permeabil- 
ity and  the  action  of  toxic  substances  in  general,  only  frag- 
ments of  which  are  cited  above,  there  have  arisen  several  more 


, 

* 

, 

. 

. 


* 


9 


or  less  definite  problems*  There  is  at  present  somewhat  of  a 
dispute  as  to  the  real  cause  of  the  impermeability  of  the  seed 
coat  of  peas*  One  group  holds  that  it  is  due  to  an  external 
layer  of  some  foreign  impervious  substance  while  a second  group 
maintains  that  the  property  of  impermeability  is  resident  in 
the  deeper  layers  of  the  seed  coat  itself  and  is  not  to  be 
isolated  therefrom.  It  is  the  purpose  of  the  following  study 
to  find  some  conclusive  evidences  upon  either  of  the  above 
contentions. 


* 


. 


10 


II  Citation  of  References. 

Arcichovskij  . , V.  - Biochemi sche  Wirkung  Hociist  Konzentrierter 
Lb  sungen. 

Biochem.  Zeitscher,  Vol.  50,  233. 

Bergtheil,  C.  and  Day,  D.  L.  - On  the  Cause  of  ’Hardness’  in 

the  Seeds  of  Indigofera  aur recta.  Annals  of  Botany,  Vol. 
21,  57. 

u 

Bokorny,  Th.  - Uber  den  Einflusz  verschiedener  Substanzen  auf 
die  Keinung  der  Pflanzen3amen.  Wachstumsforderung  durch 
Einige.  Biochem.  Zeitschr. , Vol.  50,  1. 

Brown,  A.  J.  - Semipermeable  membrane  in  Graminae.  Annals  of 
Botany,  Vol.  21,  79 

Kahlenberg,  L.  and  True,  R.  H.  - On  the  Toxic  Action  of  Dis- 
solved Salts  and  their  Electrolytic  Dissociation.  Bot- 
anical Gazette,  Vol.  22,  81. 

Kurzwelly,  W.  - Uber  die  Wiederstandsfahigkeit  trockener  pflan- 

zlicher  Organismen  gegen  giftige  Stoffe.  Jahrb.  f.  wiss.  Bot. 
1913.  Vol.  38,  291. 

Love,  H.  H.  and  Leighty,  C.  E.  - Cornell  University  Bulletin 
312.  Germanation  of  Seed  as  affected  by  sulfuric  Acid 
Treatment. 

Paine,  S.  G.  and  Saunders,  L.  M.  - Annals  of  Botany,  Vol.  32, 
175.  On  a peculiarity  Exhibited  by  the  Testa  of  Wrinkled 


Peas. 


* 


G 


f 


11 

III  Methods  and  Materials. 

Since  the  purpose  here  is  to  demonstrate  varying  amounts, 
of  permeability  of  the  seed  coat,  it  is  convenient  to  use  some 
agent  which  will  definitely  show  its  effects  upon  the  seed. 

Such  an  agent  is  found  in  Sulfuric  acid,  and  it  was  decided  to 
use  it  in  varying  concentrations  ranging  from  n/32  to  cone,  sp 
gr  1.84.  The  solutions  which  were  used  were  i\/32,  n/16,  n/8, 
n/4,  n/2,  n/l,  2n,  4n,  8n,  16n,  24n,  32n,  and  concentrated  or 
37.5  noimal  solution. 

Four  varieties  of  pea  seeds  were  used  throughout  the  ex- 
periment. These  were  Vaughan’s  Gradus,  Alaska,  Canada  Held, 
and  Black  Bye  Marrowfat.  Each  variety  was  treated  for  one  and 
two  hour  periods  by  immersion  in  each  of  the  above  concentra- 
tions. The  acid  solutions  as  well  as  the  geminating  seeds 
were  kept  at  a constant  temperature  in  the  constant  tempera- 
ture cases.  Two  complete  series  wer  run  off  - one  at  20 ’c 
and  one  at  15’ c. 

The  seeds  were  all  rinsed  in  running  tap  water  for  a 
period  of  approximately  five  minutes  following  the  aci<£  treat- 
ment. They  were  then  placed  on  the  regulation  Rag-doll  Seed 
Corn  Tester  for  germination.  The  doll  which  was  used,  however, 
was  the  modified  form  consisting  of  the  cloth  strip  wrapped 
with  a strip  of  glazed  wrapping  paper  upon  a core  of  wire 
scree/ning.  Such  an  arrangement  prevents  the  spread  of  disease 
from  one  portion  of  the  doll  to  another,  as  the  paper  is  rolled 
up  with  the  cloth  and  thus  comes  between  each  successive  layer 


i 


' t 


I I 

12 

of  the  doll.  The  seeds  on  the  doll  were  thoroughly  wetted  he- 
fore  wrapping,  and  were  wetted  also  upon  every  alternate  day 
during  the  germination  period.  Throughout  the  period  the  dolls 
v/ere  kept  in  earthenware  jars  in  the  constant  temperature  cases. 

During  the  period  of  germination  the  3ecds  were  examined 
every  alternate  day  to  determine  the  number  which  had  geminated 
during  each  period.  It  was  found  that  six  days  were  sufficient 
to  geminate  all  viable  seeds  of  all  varieties  at  both  temper- 
atures. After  six  days  in  the  dolls  the  seeds  were  removed, 
the  dolls  sterilized  by  boiling,  and  a new  lot  put  into  the 
gerrainatiors.  Lots  of  50  peas  of  each  variety  were  used  in  all 
tests  and  at  each  observation  of  the  number  of  germintated  seeds 
was  counted.  As  a criterion  of  gemination  it  was  decided  to 
use  the  rupture  of  the  seed  coat  and  a consequent  elongation 
of  the  plumule  to  the  extent  of  2 ram.  Seeds  which  showed  such 
an  elongation  but  which  appeared  to  be  no  longer  alive  were  not 
counted  as  geminated.  The  amount  of  gemination  which  is 
given  in  all  cases  is  thus  a positive  one  indicating  subsequent 
growth  of  the  seedling* 

Some  work  was  also  done  with  alcohol  to  find  out  whether 
the  permeability  was  due  to  a bloom  coat  on  the  outside  of  the 
seed,  Dor  this  purpose  samples  of  the  four  varieties  of  seeds 
used  above  were  immersed  for  two  hours  in  absolute  alcohol  at 
an  approximate  temperature  of  35’ C.  The  Seeds  were  washed  in 
running  tap  water,  dried,  and  immersed  in  concentrated  H2S04 
for  one  and  two  hours,  and  then  geminated  as  above. 


. 


. 


• 

. 


. 

. 

* 


. 

To  test  and  compare  the  results  of  acid  treatment,  check 
lots  were  run  for  both  temperatures  and  for  both  periods  of 
treatment.  The  seeds  were  soaked  in  distilled  water  for  one 
and  two  hours  and  then  treated  exactly  like  the  acid  treated 
seeds# 

It  will  be  noticed  that  in  all  cases  it  is  assumed  that 
the  rate  and  per  cent  of  germination,  and  the  appearance  and 
subsequent  growth  of  the  seedlings  are  direct  indications  of 
the  extent  to  which  the  seed  coat  is  permeable  in  each  case# 
If  the  germination  is  low  it  is  because  the  seed  coat  was 
readily  permeable  to  the  acid  and  vice  versa.  Account  was 
also  taken  in  each  case  of  the  comparative  growth  of  mold  on 
the  seeds  in  order  to  determine  if  there  is  any  relation  be- 
tween the  amount  of  this  mold  growth  and  the  acid  treatment* 


. 


- 


14 


IV  Results  and  Experimental  Data. 

The  numbers  here  expressed  indicate  percentages  of  germ- 
ination in  each  case.  They  are  given  for  periods  of  two,  four, 
and  six  days  following  acid  or  water  treatment .The  temperature 
and  the  variety  of  seed  are  indicated  at  the  top  while  the  con 
entration  of  the  solution  which  was  used  in  each  case  is  ex- 
pressed at  the  side.  The  length  of  time  of  treatment  is  also 
included  at  the  top  and  is  indicated  as  one  hour  or  two  hours. 


15 


Temperature  20  ’ C . 

Gradus  Alaska 

1 hour  2 hours  1 hour  2 hours 


2 

4 

6 

2 

4 

6 

2 

4 

6 

2 

4 

6 

cone . 

to 

94 

98 

6 

92 

94 

72 

82 

82 

48 

84 

84 

32  n. 

44 

92 

94 

4 

86 

90 

84 

100  100 

50 

94 

94 

24  n. 

32 

94 

94 

14 

90 

92 

60 

90 

96 

68 

94 

94 

16  n. 

68 

88 

92 

2 

90 

92 

96 

98 

98 

69 

100  100 

8 n. 

80 

92 

92 

16 

90 

90 

96 

98 

98 

70 

96 

96 

4 n. 

22 

76 

86 

0 

70 

70 

89 

92 

96 

18 

82 

82 

2 n. 

30 

76 

76 

0 

53 

53 

80 

86 

86 

24 

62 

62 

1 n. 

4 

78 

80 

0 

42 

50 

70 

78 

78 

34 

62 

64 

n/2. 

20 

64 

64 

0 

36 

42 

62 

42 

84 

26 

54 

62 

n/4. 

14 

62 

70 

6 

64 

60 

61 

69 

73 

44 

64 

64 

n/8. 

16 

94 

94 

12 

64 

64 

84 

96 

100 

42 

82 

84 

n/16. 

96 

86 

84 

32 

76 

80 

94 

94 

94 

70 

78 

78 

n/32. 

80 

86 

86 

58 

78 

80 

92 

94 

94 

76 

80 

84 

check 

14 

92 

92 

8 

82 

82 

80 

98 

98 

60 

94 

96 

check 

2 

84 

90 

62 

84 

88 

24 

92 

92 

98 

98 

98 

16 


Temperature  20’  C. 


Canada 

Field 

Black  Eye 

Marrowfat 

1 hour 

2 hours 

1 hour 

2 hours 

2 

4 

6 

2 

4 

6 

2 

4 

6 

2 

4 

6 

cone . 

70 

80 

80 

74 

94 

94 

46 

74 

88 

46 

82 

82 

32  n. 

82 

96 

96 

72 

88 

88 

71 

91 

96 

48 

84 

84 

24  n. 

66 

88 

96  * 

54 

96 

96 

78 

94 

94 

54 

92 

92 

16  n. 

90 

96 

96 

38 

92 

94 

82 

88 

88 

30 

92 

92 

8 n. 

88 

94 

96 

44 

90 

90 

86 

88 

88 

50 

94 

94 

4 n. 

74 

92 

92 

14 

76 

78 

48 

56 

• 

56 

4 

46 

46 

2 n. 

58 

82 

86 

8 

68  68 

36 

74 

82 

4 

30 

30 

1 n. 

44 

74 

74 

16 

50 

54 

18 

50 

52 

0 

12 

14 

n/2. 

45 

69 

73 

8 

38 

42 

32 

54 

56 

4 

20 

24 

n/4. 

40 

60 

68 

34 

64 

68 

42 

54 

60 

22 

32 

36 

n/8. 

48 

92 

92 

26 

70 

72 

• 

60 

78 

78 

30 

44 

46 

n/16. 

88 

98 

98 

46 

78 

82 

80 

82 

82 

50 

56 

58 

n/32. 

80 

86 

86 

59 

67 

71 

88 

92 

92 

58 

62 

66 

check 

64 

84 

88 

30 

65 

69 

84 

88 

92 

26 

74 

74 

check 

40 

92 

94 

56 

62 

62 

8 

7S 

> 79 

72 

74 

74 

17 


Temperature  15’  C. 
Gradus  Alaska 


1 hour 

2 hours 

1 hour 

2 hours 

2 

4 

6 

2 

4 

6 

2 

4 

6 

2 

4 

6 

cone . 

54 

94 

94 

38 

90 

90 

78 

84 

84 

38 

90 

90 

32  n. 

62 

92 

96 

0 

86 

92 

92 

100 

• 100 

20 

90 

90 

24  n. 

0 

76 

92 

4 

88 

90 

14 

98 

98 

22 

90 

90 

16  n. 

0 

78 

88 

2 

80 

86 

22 

96 

98 

18 

90 

92 

8 n. 

22 

90 

90 

0 

60 

92 

58 

96 

96 

4 

96 

ICO 

4 n. 

20 

92 

92 

0 

70 

76 

68 

90 

90 

4 

82 

CO 

CO 

2 n. 

14 

66 

78 

0 

52 

60 

66 

84 

84 

4 

80 

80 

1 n. 

10 

76 

78 

0 

42 

52 

70 

86 

88 

0 

66 

68 

n/2. 

0 

2 

76 

0 

56 

62 

0 

56 

98 

2 

70 

72 

n/4. 

0 

0 

74 

0 

24 

54 

0 

42 

94 

0 

64 

72 

n/8. 

12 

86 

92 

0 

38 

68 

72 

86 

88 

0 

84 

84 

n/16. 

20 

82 

90 

0 

30 

54 

80 

94 

98 

0 

86 

90 

check 

12 

80 

84 

12 

78 

78 

64 

98 

98 

66 

90 

92 

18 


Temperature  15*  C. 

Canada  Field  Black  Eye  Marrowfat 

1 hour  2 hours  1 hour  2 hours 


2 

4 

6 

2 

4 

6 

2 

4 

6 

2 

4 

6 

cone . 

90 

90 

90 

79 

100  100 

80 

84 

86 

48 

90 

90 

32  n. 

86 

94 

98 

42 

88 

90 

70 

92 

92 

36 

82 

90 

24  n. 

20 

94 

96 

26 

90 

90 

6 

98 

100 

30 

87 

87 

16  n. 

4 

94 

98 

14 

90 

96 

14 

78 

80 

12 

76 

84 

8 n. 

58 

86 

92 

4 

78 

94 

52 

92 

96 

0 

84 

88 

4 n. 

61 

83 

89 

8 

80 

88 

48 

62 

62 

0 

62 

64 

2 n. 

54 

84 

90 

2 

57 

67 

32 

72 

72 

0 

32 

34 

1 n. 

38 

72 

80 

2 

54 

58 

40 

56 

58 

0 

10 

16 

n/2. 

0 

24 

70 

2 

50 

56 

0 

12 

56 

0 

28 

35 

n/4. 

0 

26 

74 

0 

36 

64 

0 

20 

66 

0 

22 

26 

n/8. 

40 

82 

86 

0 

32 

52 

52 

76 

80 

0 

36 

42 

n/16. 

48 

86 

88 

0 

46 

66 

42 

68 

74 

0 

38 

50 

check 

68 

98 

98 

44 

82 

90 

48 

84 

84 

46 

78 

78 

19 


Results  in  percentages  of  germination  after  soaking  for 
two  hours  in  absolute  alcohol  at  a temperature  of  about  35* C. 
and  a subsequent  treatment  with  concentrated  Sulfuric  Acid: 

1 hour  2 hours 


2 

4 

6 

2 

4 

6 

Gradus 

0 

68 

80 

0 

72 

74 

Alaska 

12 

82 

86 

2 

96 

98 

Canada  P. 

42 

84 

86 

8 

90 

90 

Black  Eye  M. 

6 

60 

70 

4 

80 

86 

Mean 

cf  final 

20 * C.- 

Mean-4 

. germinations  for 

1 hour.  15*  C.- 

tests.  Me an -2 

a series 

■1  hour, 
tests . 

Gradus 

88 

90 

Alaska 

98 

97 

Canada  P. 

96 

82 

Black  Eye 

M.  80 

82 

20*  C.- 

2 hours. 

15 1 C.- 

■2  hours. 

Mean-3 

tests . 

1 test 

only. 

Gradus 

86 

78 

Alaska 

93 

92 

Canada  F. 

87 

90 

check  tests: 


Black  Eye  M 


82 


78 


20 

V Discussion  and  Interpretation. 

The  most  apparent  variation  in  germination  occurring  in 
these  results  is  that  one  resulting  from  a change  in  the  con- 
centration of  the  acid  solution.  Although  the  variation  se- 
cued  here  is  not  nearly  as  striking  as  that  reported  by  Arcic- 
hovskij,  it  is  nevertheless  a very  real  and  constant  variation. 
Thus  in  all  cases,  regardless  of  length  of  treatment  and  the 
variety  of  seeds  which  was  used,  the  lowest  percentage  of  germ- 
ination is  secured  by  concentrations  ranging  from  l/4  normal  to 
4 normal.  Lower  concentrations  seem  to  have  little  effect  upon 
the  percent  of  germination  while  higher  ones,  especially  up  to 
a two  hour  soaking  in  concentrated  acid,  actually  increase  the 
number  of  germinated  seeds  above  that  secured  in  the  check  germ- 
inations. There  is,  of  course,  an  occasional  exception  to  these 
general  statements,  but  these  are  very  rare  and  possess  no  more 
than  chance  significance.  A maximum  germination  is  secured  in 
nearly  all  cases  by  the  use  of  concentrations  ranging  from  8 n. 
to  concentrated  acid.  The  seeds  are  killed,  then,  principally 
by  the  medium  strength  solutions,  whereas  the  more  highly  con- 
centrated solutions  actually  enhance  germination.  Such  a phenom- 
enon is  due  largely  to  the  nature  of  the  seed  coat  of  the  peas. 

This  coat  although  not  selectively  permeable,  and  hence  ex- 
ercising no  semi-permeable  activity  in  regard  to  the  acid  sol- 
utions, is  nevertheless  very  impermeable  to  the  acid.  Especially 
is  this  the  case  with  dry  seed  coats.  When  they  once  imbibe  water 
from  the  surrounding  solution  the  acid  has  a ready  access  to  the 
interior  of  the  seed,  an  d a lowering  in  the  germination  occurs. 
Such  an  explanation  seems  to  fit  in  quite  readily  with  the  above 
results.  A maximum  amount  of  water  is  imbibed  from  concentrations 


. 


21 


below  8 normal,  and  the  amount  of  acidin  such  solutions  is  suf- 
ficient to  materially  cut  down  the  germination  until  a concen- 
tration of  1/4  normal  is  reached.  Below  this  point  the  amount  of 
acid  is  so  small  that  there  i s no  apparent  decrease  in  the  germ- 
ination. 

In  close  relation  to  the  variation  of  the  percentage  of  ger- 
mination with  a change  in  the  concentration  of  the  solution  is 
the  change  in  the  rate  of  germination.  The  results  show  a very 
striking  temperature  effect  in  this  connection.  At  15’ C.  the  rate 
of  germination  is  in  nearly  all  cases  materially  slower  than  at 
20* C.  The  rate  of  germination  is  also  influenced  by  the  length 
of  the  period  of  treatment.  A two  hour  exoosure  to  acid  usually 
results  in  a materially  slower  germination  rate  than  that  secured 
by  a one  hour  treatment.  Furthermore,  the  rate  is  slowest  where 
the  germination  percentage  is  lowest.  Such  a slowing  in  the  rate 
is  usually  manifested  by  a retarded  beginning  of  the  germination 
process . 

It  is  also  apparent  that  the  length  of  the  time  of  treatment 
also  has  a very  direct  influence.  Except  in  a very  small  number  ol 
cases,  there  is  a lower  percentage  of  germination  for  the  two 
hour  treatment  than  for  the  one  hour  exposure.  This  is  doubtless- 
ly due  to  the  greater  length  of  time  during  which  the  acid  acts 
upon  the  seed  coat  in  case  of  the  longer  periods  of  treatment, 
as  well  as  the  lengthened  time  for  the  imbibition  of  water  in 
such  a case.  In  check  germinations  there  occurs  no  such  differ, 

ence;  in  fact,  the  one  huur  soaking  usually  gives  a higher  per- 
centage of  germination  than  the  two  hour  treatment. 

In  all  cases,  it  will  be  noticed  that  the  percent  of  germ- 
ination  is  higher  for  the  treatment  at  15 'C.  than  at  20*0. 


, 


22 

Although  there  is  a similar  variation  in  each  temperature  series, 
according  to  the  strength  of  the  solution,  the  percent,  germin- 
ation for  germination,  is  always  higher  for  the  15’  series.  This 
fact  is  due  directly  to  the  lessened  chemical  activity  of  tve  sol- 
utions at  the  lov/er  temperature. 

Another  very  striking  variation,  and  one  to  which  few  of  the 
earlier  investigators  paid  much  heed,  is  the  one  occurring  between 
the  different  varieties.  This  variation  is  quite  marked  and  is 
constant  throughout  all  concentrations  and  times  of  treatment,  al- 
though it  is  not  nearly  as  apparent  with  the  higher  and  lower 
concentrations  as  it  is  in  the  dase  of  the  most  poisonous  ones. 
With  few  exceptions  the  order  of  the  percent  of  germination  from 
highest  to  lowest  is  as  follows:  Alaska,  Gradus,  Canada  Field, 
and  Black  Eye  Marrowfat.  Although  the  absolute  percentages  vary 
for  different  concentrations,  times,  and  temperatures,  their  rel- 
ative rank  always  remains  the  same.  Thus,  although  the  lowest 
germination  percent  ranges  from  1/4  normal  to  2 normal,  it  always 
occurs  in  the  casw  of  the  Black  Eye  Marrowfat  variety.  This  is 
an  excellent  demonstration  of  the  varietal  as  well  as  individual 
variation  of  the  seed  coat  characteristics. 

The  growth  of  mold  was  also  observed  throughout  the  alcove 
experimentation.  The  greatest  variation  in  this  regard  came  in 
relation  to  the  number  of  dead  peas.  Mold  was  not  observed  grow- 
inh  on  living  peas  in  any  case,  and  the  amount  hence  always  var- 
ied inversely  with  the  number  of  germinated  peas.  Although  there 
was  a very  profuse  growth  of  mold  in  such  cases  at  20’,  the  am- 
ount of  mold  at  15’  was  very  slight  even  in  the  cases  of  minim- 
um germination.  Bacterial  contamination,  as  evidence  by  sliminess 

of  the  seed  coat,  was  not  materially  decreased  toy  the  lowering 

in  tifimpprflt.nrfl.  


‘ 


23 


Another  very  interesting  occurrence  is  the  process  of  pit- 
ting of  the  seed  coat.  This  pitting  occurs  in  the  use  of  concen- 
trations ranging  from  24  normal  to  concentrated  acid,  an  d is  most 
marked  with  the  latter  treatment.  It  is  interesting  to  note  that 
the  amount  of  tvis  pitting  is  quite  closely  correlated  with  the 
percentage  of  germination.  The  extent  of  the  pitting  seems  to  he 
directly  indicative  of  the  extent  of  acid  injury.  There  is  also 
a distinct  individual  difference  in  the  amount  of  pitting.  Some 
seeds  are  exceedingly  pitted  and  almost  charred  by  soaking  in  the 
concentrated  acid,  while  others  withstand  even  a two  hour  expos- 
ure with  little  apparent  injury  to  the  seed  coat. 

Finally,  there  remains  to  be  discussed  the  very  interesting 
results  of  the  work  with  alcohol. From  the  results,  it  will  be 
observed  that  a treatment  of  two  hours  with  absolute  alcohol  at 
a temperature  of  35* C.,  followed  by  a subsequent  soaking  of  one 
to  two  hours  in  concentrated  acid,  resulted  in  percentages  of  ger- 
mination not  very  far  removed  from  those  attained  by  the  treat- 
ment with  concentrated  Sulfuric  Acid  without  presoaking  in  alco- 
hol. The  variation  between  the  two  is  not  much  more  than  that 
whidh  occurs  between  any  duplicate  series.  Not  only  is  the  ger- 
mination percentage  lowered  in  only  a few  cases,  and  then  but 
slightly,  but  in  an  equal  number  of  cases  the  percentage  of  ger- 
mination is  actually  increased  after  the  alcohol  treatment. 

It  would  seem  on  the  basis  of  t ese  results,  that  the  con- 
clusion of  Paine  and  Saunders  in  regard  to  the  cause  of  the  im- 
permeability of  the  seed  coat  of  peas,  does  not  hold  for  the  var- 
ieties which  were  used  in  this  work.  Neitherwas  it  possible  to 
establish  any  certain  correlation  between  thd  particular  nature 
of  the  seed  coat  in  each  variety  with  its  percentage  of  germin- 


* 
* 


24 


ation.  Of  the  four  varieties  which  were  used,  Gradus  had  the  most 
wrinkled  seed  coat,  while  Alaska  came  second  in  thid  regard.  The 
coats  of  Can  ada  Field  and  Black  Eye  Marrowfat  are  smooth.  There 
is  no  apparent  relation  between  these  facts  and  the  results  of  the 
experiment-either  in  the  case  of  check,  straightacid,  or  alcohol 
plus  acid  treatment.  It  was  furthermore  impossible  to  establish 
any  correlation  between  the  amount  of  pitting,  spoken  of  above, 
and  the  nature  of  the  seed  coat  with  reference  to  smoothness.  It 
must  therefore  be  concluded  that  the  property  which  renders  the 
seed  coat  of  peas  relatively  impermeable  to  the  entrance  of  acid, 
located  within  the  structure  of  the  seed  coat,  and  is  not  an  ex- 
ternal coat  of  wax,  bloom,  or  other  impervious  material. 


25 


VI  Conclusions. 

1.  The  percent  of  germination  is  lowest  for  medium  concen- 
trations, but  this  is  due  rather  to  an  increased  permeability  of 
the  seed  coat  than  to  ary  intrinsic  quality  of  greater  poison- 
ousness on  the  part  of  such  concentrations. 

2.  The  percent  of  germination  is  higher  with  treatment  at 
15'  than  at  20' C.,  and  the  rate  of  germination  is  retarded  by 
a decrease  in  the  temperature. 

3.  An  increase  in  the  length  of  treatment  with  acid  results 
in  a lowering  of  the  percent  of  germination. 

4.  There  exists  a marked  variation  in  germination  between 
the  varieties  of  peas;  the  order  from  highest  to  lowest  being: 
Alaska,  Gradus,  Canada  Field,  and  Black  Eye  Marrowfat.  Individ- 
ual variation  is  also  very  apparent. 

5.  Since  presoaking  for  several  hours  with  warm  alcohol  seem: 
to  have  no  significant  effect  on  the  percent  of  germination,  it 

is  concluded  that  the  impermeable  property  is  not  due  to  an  ex- 
ternal coating  of  bloom  or  wax. 


26 


VII  Figures. 

These  figures  are  constructed  from  the  data  cited  under 
the  heading  of  Results  and  Experimental  Data.  They  are  in- 
tended to  show  in  graphic  form  the  respective  curves  of  var- 
iation for  the  varieties  of  seed  for  different  lengths  of 
treatment  in  the  consecutive  concentrations  of  solutions. 


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